ABSTRACT: Project 1 ? Crystallography and NMR-guided Generation of Fungal Calcineurin Inhibitors The conserved phosphatase calcineurin is broadly necessary for invasive fungal disease. FDA-approved calcineurin inhibitors (FK506, cyclosporine A (CsA)) are active against invasive fungal pathogens, but they also suppress host immunity. Our preliminary studies document proof of principle compounds that have antifungal activity in vitro and in vivo and are non-immunosuppressive. The goals of this proposal are to identify fungal- specific targets in the calcineurin pathway via fungal-mammalian structural differences and develop novel inhibitors. The central hypothesis is that a structural biological approach through crystallography and NMR spectroscopy will define novel and targetable fungal-specific areas in the calcineurin circuit critical for fungal pathogenesis. We hypothesize that structures of the calcineurin AB complex, coupled with calmodulin and the immunophilin complexes (cyclophilin A-CsA, FKBP12-FK506), will reveal novel fungal-specific targets for inhibition. Our successful preliminary studies have already solved structures of the calcineurin AB complex for Candida albicans and Aspergillus fumigatus. To extend maximal clinical breadth, this proposal will now focus on Cryptococcus neoformans/gattii, Candida glabrata, and Mucor circinelloides. Cryptococcal meningitis is the most common etiology of fungal central nervous system disease worldwide, Candida glabrata is emerging as both an azole- and echinocandin-resistant species, and mucormycosis is the 3rd most common invasive fungal infection in all transplant recipients. We will solve structures for the calcineurin heterodimer alone and complexed with FKBP12-FK506/analogs and cyclophilin A-CsA/analogs for these three pathogens. The multiple molecular views provided will allow identification of sites for chemical targeting that are distinct between human and fungal calcineurin complexes. Protein regions that are dynamic or resist crystallization will be structurally characterized by NMR. Putative inhibitory domains will be validated via genetic and biochemical assays, utilizing site-directed mutagenesis of key contact and surface residues to examine structural stability, fungal phenotype, and drug action/resistance. Non-immunosuppressive fungal-specific calcineurin inhibitors will be generated by: 1) screening libraries of CsA analogs (Scynexis, Novartis), FK506 analogs (Amplyx Pharmaceuticals), and FK506-tripeptide conjugates (Jun Liu, Johns Hopkins) to provide the structural basis to pursue novel chemical modifications based on known drug platforms; and 2) conducting medicinal chemistry in collaboration with investigators at Amplyx and Scynexis to alter analogs based on our screening/structural results. This will provide insight to produce second-generation inhibitors optimized for potentiation of antifungal activity and abrogation of immunosuppression by capitalizing on unique structural differences between the host and fungal enzymes. Non-immunosuppressive calcineurin inhibitors will advance to the Inhibitor Testing Core for antifungal activity screening, cell-based toxicity assays, and animal model efficacy testing. Results will feedback to this Project for any additional medicinal chemistry modifications.